EP0281356A1 - Segment d'ADN conférant transduction à haute fréquence de vecteurs d'ADN recombinants - Google Patents

Segment d'ADN conférant transduction à haute fréquence de vecteurs d'ADN recombinants Download PDF

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EP0281356A1
EP0281356A1 EP88301763A EP88301763A EP0281356A1 EP 0281356 A1 EP0281356 A1 EP 0281356A1 EP 88301763 A EP88301763 A EP 88301763A EP 88301763 A EP88301763 A EP 88301763A EP 0281356 A1 EP0281356 A1 EP 0281356A1
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phage
dna
plasmid
streptomyces
hft
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EP0281356B1 (fr
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Richard Henry Baltz
Margaret Ann Mchenney
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces

Definitions

  • the present invention relates to transducible vectors that utilize a segment of phage FP43 DNA that enables transduction of the vectors by phage FP43.
  • Phage FP43 does not have cohesive ends, so the trans­duction system of the present invention differs from the ⁇ system for E . coli , the SP02 system for Bacillus subtilis , and the R4 system for S . lividans .
  • the present invention also relates to a method of trans­ferring DNA into a host cell that involves the use of transducing vectors. The method allows efficient transfer of DNA between many species of Streptomyces and transduction of several other general of actinomycetes.
  • restriction fragment size may vary somewhat from calcu­lated size based on map distances.
  • restriction enzymes such as MboI , only certain cut sites are shown for convenience.
  • the present invention relates to vectors and methods for a phage-mediated transduction system not only for Streptomyces but also for other organisms throughout the Actinomycetales family, such as Chainia , Saccharopolyspora , and Streptoverticillium .
  • the transduction system utilizes a segment of DNA from bacteriophage FP43 cloned into illustrate phasmids pIJ702 (see Katz et al ., 1982, J. Gen. Microbiol. 192: 2703-2714) and pMT660 (see Birch and Cullum, 1985, J. Gen. Microbiol. 131: 1299-1303).
  • FP43 is a temperate bacteriophage with broad host specificity for Streptomyces spp.
  • the present invention provides transducible cloning vectors for use in Streptomyces and other host cells.
  • the development and exploitation of recombinant DNA technology in Streptomyces depends upon the avail­ability of suitable cloning vectors. This development has been somewhat retarded by the low number of suitable vectors presently available for use in Streptomyces and other antibiotic-producing organisms and the difficul­ties encountered in developing protoplast transformation procedures.
  • the present invention is useful and especially important in that it expands the number of vectors and hosts suitable for such use.
  • the vectors of the present invention are particularly useful, because the vectors are small, versatile, and can be transformed and selected in all Streptomyces species and many other heretofore untrans­formable organisms. Streptomyces provides over half of the clinically important antibiotics and thus is a commercially significant group.
  • the present invention provides new and useful cloning systems and vectors for this industrially important group and allows for the cloning of genes both for increasing the yields of known antibiotics and also for producing new antibiotics and antibiotic derivatives.
  • Antibiotic - a substance produced by a micro-­organism which, either naturally or with limited modi­fication, will inhibit the growth of or kill another microorganism or eukaryotic cell.
  • Antibiotic Biosynthetic Gene - a DNA segment that encodes an enzymatic or other activity that is necessary in the process of converting primary metabo­lites into antibiotics.
  • Antibiotic Biosynthetic Pathway the entire set of antibiotic biosynthetic genes necessary for the process of converting primary metabolites into anti­biotics.
  • Antibiotic Resistance-Conferring Gene a DNA segment that encodes an enzymatic or other activity that confers resistance to an antibiotic.
  • Genomic Library a set of recombinant DNA cloning vectors into which segments of DNA, comprising substantially all of the DNA of a particular organism or phage, have been cloned.
  • hft - high frequency transduction also used to denote a segment of phage DNA that confers high frequency transducibility to a vector.
  • Infection the process of phage replication, wherein the phage attaches to and injects its DNA into the host cell, which then supports phage replication and maturation and ultimately release of phage particles through lysis.
  • Recombinant DNA Cloning Vector--any auto­nomously replicating or integrating agent, including, but not limited to, plasmids, comprising a DNA molecule to which one or more additional DNA segments can be or have been added.
  • Restriction Fragment--any linear DNA molecule generated by the action of one or more restriction enzymes generated by the action of one or more restriction enzymes.
  • Selectable Marker - a segment of DNA incor­porated into a recombinant DNA vector that allows for the identification of cells containing the vector, whether freely replicating or integrated.
  • Selectable markers include antibiotic resistance-conferring genes and other genes such as the tyrosinase or ⁇ -galactosidase genes.
  • Sensitive Host Cell--a host cell that cannot grow in the presence of a given antibiotic without a DNA segment that provides resistance thereto.
  • TcR - the tetracycline-resistant phenotype or gene conferring same.
  • Transductant - a recipient host cell that has undergone transduction
  • Transformant - a recipient host cell that has undergone transformation.
  • the present invention provides a method for isolating sequences of actinophage (a phage that infects organisms in the family Actinomycetales DNA that confer high frequency transduction to recombinant vectors.
  • actinophage a phage that infects organisms in the family Actinomycetales DNA that confer high frequency transduction to recombinant vectors.
  • hft sequences can be isolated by: (1) making a genomic library of the actinophage DNA; (2) transforming the genomic library into a host that supports actinophage infection; (3) infecting the cell transformed in step (2) with the actinophage and collecting the resulting lysate; (4) using the lysate to transduce an organsim of the family Actinomycetales ; and (5) isolating the vectors that transduce at high frequency in step (4).
  • This method is especially preferred for isolating hft sequences from Streptomyces phages. The method is illustrated by demonstrating the isolation of
  • Vectors constructed with an hft sequence isolated by the method of the present invention are especially useful in a method of transferring DNA encoding a useful substance to an actinomycetes.
  • This method comprises transducing an actinomycetes with a recombinant DNA vector that comprises: (1) an hft squence; (2) DNA encoding a useful substance; (3) a plasmid origin of replication; and (4) a selectable marker.
  • the DNA molecules can encode such useful substances as antibiotic biosynthetic enzymes, intact antibiotic biosynthetic genes or pathways, and pharmacologically active substances, such as growth hormone, insulin, interferon, and the like.
  • the present invention also comprises a DNA segment of bacteriophage FP43 that can be used to construct recombinant DNA expresion vectors that are transducible at high frequency in a wide variety of organisms.
  • Bacteriophage FP43 can be isolated from Streptomyces griseofuscus (FP43) by the procedure described in Example 1.
  • S . griseofuscus (FP43) contains lysogenized FP43 phage and is available from the Agricultural Research Service, Northern Regional Research Center (NRRL), Peoria, IL 61604, under the accession number NRRL 18184.
  • the segment of bacteriophage FP43 DNA that confers high frequency transducibility can be isolated readily and used to construct vectors of the present invention.
  • Illustrative vector pRHB101 was constructed by inserting the ⁇ 7.8 kb Sph I restriction fragment of phage FP43 into Sph I-digested plasmid pIJ702.
  • the ⁇ 7.8 kb Sph I restriction fragment of phage FP43 comprises the hft sequence.
  • Plasmid pIJ702 is a multicopy plasmid about 5.8 kb in size that has broad host specificity for Streptomyces (see Acebal et al ., 1986, FEMS Microbiol. Lett. 35: 79-82; Katz et al ., 1982, J. Gen. Microbiol. 192: 2703-2714; Lampel and Strohl, 1986, Appl. Environ. Microbiol. 51: 126-131; and Matsushima and Baltz, 1985, J. Bacteriol.
  • Plasmid pIJ702 was derived from the multicopy, broad host range plasmid pIJ101 (Kieser et al ., 1982, Mol Gen. Genet. 185: 223-238) and can be obtained from the American Type Culture Collection, Rockville, MD 20852, under the accession number ATCC 39155.
  • a restriction site and function map of plasmid pIJ702 is presented in Figure 1 of the accompanying drawings.
  • Plasmid pRHB101 from the ⁇ 7.8 kb Sph I restriction fragment of phage FP43 and Sph I-digested plasmid pIJ702 is described in Example 2, below.
  • a restriction site and function map of plasmid pRHB101 is presented in Figure 2 of the accompanying drawings.
  • Plasmid pMT660 is a mutant of plasmid pIJ702 that is temperature-sensitive for replication in Streptomyces lividans (see Birch and Cullum, 1985, J. Gen. Microbiol. 131: 1299-1303).
  • a restriction site and function map of plasmid pRHB106 is presented in Figure 3 of the accompanying drawings. Plasmid pRHB106 can be obtained from S . griseofuscus C581/pRHB106 in substantial accordance with the pro­cedure of Example 3. S . griseofuscus C581/pRHB106 can be obtained from the Northern Regional Research Center under the accession number NRRL 18183.
  • the above-described illustrative vectors comprise the Streptomyces replicon derived from plasmid pIJ702.
  • the present invention is not limited to the use of any particular replicon, for the trans­duction system of the present invention can be used with a variety of Streptomyces replicons or replicons derived from other organisms.
  • Table I is an illustrative, but not comprehensive, listing of Streptomyces plasmids from which Streptomyces replicons can be obtained.
  • Those skilled in the art recognize that, so long as the replicon function is not disrupted, all or part of the plasmids can be used to construct vectors that contain the hft sequence of the present invention.
  • the plasmid-­containing host and depository accession number are also listed in Table 1.
  • Restriction fragments used to construct vectors illustrative of the present invention can be conventionally modified to facilitate ligation.
  • molecular linkers can be provided to a particu­lar hft -containing restriction fragment or to DNA comprising vector replication functions.
  • specific sites for subsequent ligation can be conveniently constructed.
  • the various hft -containing restriction fragments, origin of replication, or other sequences of a given vector can be modified by adding, eliminating, or substituting certain nucleotides to alter characteristics and to provide a variety of restriction sites for ligation of DNA.
  • nucleotide chemistry and the genetic code and thus which nucleotides are interchangeable and which DNA modifications are desirable for a specific purpose.
  • hft -­containing restriction fragment is not limited to a particular position on a cloning vector, as long as critical, vector-controlled functions are not disrupted.
  • Those skilled in the art understand or can readily determine which sites on a vector are advantageous for the ligation or insertion of a particular hft -containing restriction fragment.
  • the present invention limited to the use of the ⁇ 7.8 kb Sph I restriction fragment of phage FP43 to confer transducibility to a particular vector.
  • the ⁇ 7.8 kb Sph I restriction fragment of phage FP43 is believed to comprise an origin of replication that causes a plasmid containing the origin to replicate by a rolling-circle mechanism, similar to the T4 system in E . coli (see Kreuger et al ., 1985, Proc. Natl. Acad. Sci. USA 82: 3345-3349).
  • Linear concatemers of a plasmid containing the FP43 origin of replication when exposed to intracellular phage FP43 gene products, are packaged by a headful mechanism; and resulting "packages" or particles can inject DNA into cells, and the DNA is recircularized in the transductants.
  • ⁇ 7.8 kb Because the origin of replication of phage FP43 is smaller than ⁇ 7.8 kb, the entire ⁇ 7.8 kb Sph I restriction fragment of phage FP43 is not required to confer transducibility.
  • Other restriction fragments of phage FP43 that comprise the origin of replication of phage FP43 can be used in place of the ⁇ 7.8 kb Sph I restriction fragment for purposes of the present trans­duction system.
  • larger restriction fragments of phage FP43 that comprise the ⁇ 7.8 kb Sph I restriction fragment of phage FP43 can also be used for purposes of the present transduction system.
  • phage FP43 DNA was digested with restriction endonuclease Sph I, and the DNA fragments were cloned into the Sph I site located in the mel gene of both plasmids pIJ702 and pMT660.
  • Inactiviation of the mel gene occurs when large DNA fragments are inserted into the Sph I site of either plasmid pIJ702 or pMT660. This inactivation is readily detectable, for the mel gene product converts tyrosine into a dark-colored compound. Transformants that contain an intact mel gene thus appear much darker than transformants that do not contain an intact mel gene on tyrosine-containing plates.
  • Plasmid DNA was isolated from the transformants to confirm the presence of DNA inserts. Plasmids containing inserts of FP43 DNA were transformed into S . griseofuscus and S . ambofaciens , and FP43 lysates were prepared on the transformants. The lysates were used to transduce plasmid into wild-type S . griseofuscus and S.
  • ambofaciens, , and thiostrepton-­resistant transductants were scored.
  • the ⁇ 7.8 kb Sph I restriction fragment of FP43 (designated hft for high frequency transduction) caused at least a 105-fold increase, as compared with plasmid pIJ702 with no insert, in transduction in S . griseofuscus , as dem­onstrated in Table 2, below.
  • the average transduction frequency obtained was about 10 ⁇ 4 per plaque-forming unit. No transduction ( ⁇ 2.2 X 10 ⁇ 9 per PFU) was observed with plasmid pIJ702 containing no inserts. As indicated in Table 2, other plasmids containing inserts of FP43 DNA gave trans­duction frequencies only slightly higher than observed for plasmid pIJ702. Thus, a generalized enhancement of transduction by cloning random fragments of FP43 was not observed, so this system differs from the ⁇ 105 and SP02 transduction systems studied in Bacillus and the P22 system studied in Salmonella .
  • the transduction system of the present invention also differs from other systems, because the hft segment can be transduced into most species of Streptomyces .
  • Lysates of phage FP43 prepared on S . griseofuscus containing plasmid pRHB101 and S . griseofuscus containing plasmid pRHB106 were used to transduce many other species of Streptomyces , including both species that do and do not support plaque-formation by FP43.
  • some express restrcition endonucleases that cleave FP43 DNA see Cox and Baltz, 1984 J. Bacteriol. 159: 499-504).
  • Streptomyces lavendulae was not transduced to thio­strepton resistance by FP43.
  • the results of these transductions are presented in Table 3.
  • the species transduced included S albus P, which produces Sal PI, and isoschizomer of Pst I. Plasmids pRHB101 and pRHB106 contain one site for Pst I, so the present system provides a means of overcoming such restriction systems. Table 3 also shows that the transduction system of the present invention can even be used to transform organisms resistant to phage FP43 infection. Of the 11 Streptomyces species that do not support plaque-formation by FP43, seven can be trans­duced to thiostrepton resistance by FP43. Therefore, FP43 clearly attaches and injects DNA into these species, as predicted from the host range analysis (see Cox and Baltz, 1984, J. Bacteriol. 159: 499-504).
  • Phage FP43 is probably restricted to a much greater extent in these species than plasmids pRHB101 and pRHB106. For instance, FP43 has many sites for Sph I produced by S. phaeochromogenes and is completely restricted, whereas plasmid pRHB101 has only two sites for Sph I and trans­duces at low but detectable frequency of 8.5 x 10 ⁇ 9 per plaque-forming unit.
  • Phage FP43 does not form plaques on S . albus G, S . acromogenes , or S . phaeochromogenes , the producers of restriction endonucleases Sal I, Sac I, and Sph I, respectively; FP43 DNA has many sites for all three enzymes. FP43 forms plaques on S . albus P and S . tubercidicus , the producers of Sal PI ( Pst I) and Stu I, respectively; FP43 DNA has no Pst I or Stu I sites. These observations suggest that FP43 might attach to and inject DNA into most, if not all, species of Strepto - myces .
  • FP43 packaged a plasmid containing a relatively small number of restriction sites, it might transduce that plasmid into a strain that is highly restricting for EP43.
  • Streptomyces griseofuscus S . ambofaciens , S . lividans , S . parvulus , and S . albus J1074 are relatively non-­restricting; S . albus G ( Sal I), S . lavendulae ( Sla I), S . phaeochromogenes ( Sph I), S . acromogenes ( Sac I, Sac II, and S .
  • tubercidicus ( Stu I) produce well characterized restriction systems (shown in parenthetical remark following the strain name); and strains S . aureofaciens , S . cirratus , S . coelicolor , S . griseus , S . narbonensis , S . thermotolerans , S . venezuelae , and S . macrosporeus are suspected of producing restriction systems.
  • the transduction system of the invention works well in 13 of the 14 strains tested that FP43 forms plaques on and works well in 7 of 11 strains that FP43 does not form plaques on.
  • S . albus G produces Sal I, an enzyme that cuts plasmid pRHB101 five times. Only about 20% of the strains tested produce restriction systems that are not readily bypassed.
  • S . albus P produces Sal PI, and isoschizomer of Pst I that cuts plasmid pRHB101 one time, and S . phaeochromogenes produces Sph I, which cuts plasmit pRHB101 two times.
  • the relative transduction frequency was improved 100-fold by preparing the trans­ducing lysate in S . albus P. It appears, therefore, that the plasmid was efficiently modified for the Sal PI restriction system after replication in S . albus P.
  • This procedure often referred to as "laundering" the DNA, can be used in other species to improve trans­duction efficiencies and is believed to work by a mechanism involving modification (i.e., methylation) of the transducing DNA by the "laundering" organism ( S . albus P in the procedure above).
  • This laundering procedure was carried out by transducing S . albus P to thiostrepton resistance using an FP43 lysate prepared on S . griseofuscus containing plasmid pRHB101. A subsequent FP43 lysate was prepared on S . albus P containing plasmid pRHB101.
  • the two transducing lysates were compared for their relative abilities to transduce S . griseofuscus and S . albus P strains not containing plasmid.
  • Table 4 shows that the lysate prepared on S . griseofuscus transduced S . albus P to thiostrepton resistance about 15% as efficiently as it transduced native S . griseofuscus .
  • the lysate prepared on S . albus P however, transduced both species at about equal efficiencies. Thus passageing the plasmid through the restricting host increased the relative transduction on the restricting host by about 100-fold.
  • Lysates of phage FP43 prepared on S . griseofuscus /pRHB101 were mixed with cells prepared from several species of different actinomycete genera, and plaque-formation and transduction was scored. The results are presented in Table 5, below.
  • FP43 caused plaque-formation on only Strepto­verticillium oliverticulli and Chainia minutisclerotica .
  • transduction was successful not only in Strepto­verticillium and Chainia , but also in Saccharopolyspora .
  • FP43 can attach and inject plasmid DNA into a variety of actinomycete genera and that plasmid pRHB101 can establish and replicate in many different genera.
  • the transduction system described here provides a very powerful technique to move cloned genes between a variety of Streptomyces species and into at least several other actinomycete genera and eliminates the need to develop transformation systems for each species of interest. This should accelerate the applications of recombinant DNA technology in actinomycetes to produce novel or hybrid antibiotics.
  • the wide host range of the transduction vectors of the preseent invention provides an enormous advantage over protoplast-transformation in experiments and procedures for transferring genes from one organism to another. Because this transduction system works in such a diversity of organisms, the optimal conditions for transducing one organism may differ from the optimal conditions for transforming a different organism.
  • One condition that can be optimized is the multiplicity of infection (m.o.i.).
  • the hft sequence of phage FP43 is incorporated into a recom­binant DNA expression vector. That vector (i.e., plasmid pRHB101 or plasmid pRHB106) is then transformed, by conventional protoplast-transformation procedures, into an organism, such as Streptomyces griseofuscus C581 (ATCC 23916). The resulting transformants are then infected with phage FP43. Upon phage infection, plasmids containing the hft sequence are replicated and packaged into phage heads. The resulting lysate con­tains noninfective particles that have packaged the hft -containing plasmid DNA.
  • the resulting lysate also contains, however, infective phage particles that have packaged the phage FP43 genome. These "wild-type” infective particles can cause lysis, and are thus referred to as “plaque-­forming units” or "PFU", of the recipient host cells when the lysate is used in a subsequent transduction.
  • PFU plaque-­forming units
  • Figure 4 shows several typical responses in transduction frequency to increased phage concentration for species that are hosts for FP43 and for species that are not. While the efficiencies of transduction observed in different species of Streptomyces vary from about 10 ⁇ 8 to 10 ⁇ 4 per PFU, transduction efficiency has little bearing on the maximum number of transductants obtainable on a transduction plate. The highest trans­duction frequencies are obtained on nonrestricting or marginally restricting hosts that are susceptible to lysis by FP43.
  • FP43 forms plaques on Streptomyces albus P at an efficiency-of-plating (EOP) of about 10% relative to the maximum EOP observed on S . griseofuscus .
  • EOP efficiency-of-plating
  • the frequency of transductants of S . albus P increased linearly with increasing PFU to about 3 x 107 PFU per plate and then plateaued. However, the relative efficiency of transduction per PFU was about 100-fold lower than that observed on S . griseofuscus .
  • the present invention provides a variety of means for overcoming a host cell's endogenous DNA restriction/modification system. Elevated temperature can often inhibit secondary metabolic functions such as antibiotic production and sporulation in Streptomyces . Many restriction/modification systems may be regulated in a similar way, so growth of cells at elevated tem­perature might result in decreased expression of restriction and increased transduction in some restricting strains.
  • S . griseofuscus , S . albus P, S . phaeochromogenes , S . thermotolerans , and S kentuckense were grown at 29°C and 39°C before transduction and incubated at 29°, 34° or 42°C after transduction. Table 6 below details the results of this experiment.
  • the data demonstrates that this transduction system can be readily manipulated by changing cell growth parameters to optimize transduction for particular species.
  • the recombinant DNA cloning vectors of the present invention have broad utility and help fill the need for suitable cloning vehicles for use in Strepto­myces and related organisms. Moreover, the ability of the present vectors to confer antibiotic resistance pro­vides a functional means for selecting transductants. This is important because of the practical necessity for determining and selecting the particular cells that have acquired vector DNA in a transformation procedure.
  • Additional DNA segments that lack functional tests for their presence can also be inserted into the present vectors, and transductants containing the non­selectable DNA can be isolated by selection for carbo­mycin resistance.
  • Such non-selectable DNA segments can be inserted at any site, except within regions necessary for transduction or within the antibiotic resistance-­conferring gene used for selection, and include, but are not limited to, genes that specify antibiotic modifi­cation enzymes and regulatory genes of all types.
  • the vectors of the present invention are also useful for ensuring that linked DNA segments are stably maintained in host cells over many generations. These genes or DNA frgements are maintained by exposing the transductants to selective pressure based upon the markers (i.e., an antibiotic resistance-conferring gene) present on the vector. Therefore, transductants that lose the vector cannot grow and are eliminated from the culture. Thus, the vectors of the present invention can stabilize and maintain DNA sequences of interest.
  • the cloning vectors and transductants of the present invention provide for the cloning of genes to improve yields of various products that are currently produced in Streptomyces and related cells. Examples of such products include, but are not limited to, Strepto­mycin, Tylosin, Cephalosporins, Actaplanin, Narasin, Monensin, Tobramycin, Erythromycin, and the like.
  • the present invention also provides selectable vectors that are useful for cloning, characterizing, and reconstruct­ing DNA sequences that code for: commercially important proteins such as, for example, human insulin, human proinsulin, glucagon, interferon and the like; enzymatic functions in metabolic pathways leading to commercially important processes and compounds; or control elements that improve gene expression.
  • DNA se­quences also include, but are not limited to, DNA that codes for enzymes that catalyze synthesis of derivatized antibiotics such as, for example, Streptomycin, Cephalo­sporin, Tylosin, Actaplanin, Narasin, Monensin and Erythromycin derivatives, or for enzymes that mediate and increase bioproduction of antibiotics or other products.
  • derivatized antibiotics such as, for example, Streptomycin, Cephalo­sporin, Tylosin, Actaplanin, Narasin, Monensin and Erythromycin derivatives.
  • the capability for isolating and using such DNA segments allows for increasing the yield and avail­ability of antibiotics that are produced by Streptomyces and related organisms.
  • Streptomyces can be cultured in a number of ways using any of several different media.
  • Preferred carbohydrate sources in a culture medium include, for example, molasses, glucose, dextrin, and glycerol.
  • Nitrogen sources include, for example, soy flour, amino acid mixtures, and peptones.
  • Nutrient inorganic salts are also incorporated and include the customary salts capable of yielding sodium, potassium, ammonium, calcium, phosphate, chloride, sulfate, and like ions.
  • essential trace elements are also added. Such trace elements are commonly supplied as impurities incidental to the addition of other constituents of the medium.
  • Streptomyces is grown under aerobic culture conditions over a relatively wide pH range of about 5 to 9 at temperatures ranging from about 15° to 40°C.
  • a culture medium For plasmid stability and maintenance, it is desirable to start with a culture medium at a pH of about 7.2 and maintain a culture temperature of about 30°C.
  • Phage lysates and DNA were prepared in sub­stantial accordance with the procedure described in Cox and Baltz, 1984, J. Bacteriol. 159: 499-504.
  • a lyophil­ized culture of Streptomyces griseofuscus C581(FP43) is obtained from the Northern Regional Research Center (NRRL), Agricultural Research Service, Peoria, IL 61604 under the accession number NRRL 18184.
  • the lyophil­ized culture is used to inoculate 10 ml of NC broth; the culture is then incubated at 29°C in a gyrotory incu­bator overnight ( ⁇ 16 hours).
  • the culture is centrifuged to remove the cells and cellular debris; the supernatant was then passed through a 0.45 ⁇ filter, and the filtrate was saved and contained phage FP43 particles.
  • a lyophil of Streptomyces griseofuscus C581 is obtained from the American Type Culture Collection (ATCC), Rockville, MD 20852 under the accession number ATCC 23916.
  • the lyophilized culture is used to inoculate 10 ml of TSB broth (Baltimore Biological Laboratories, Inc. (BBL), P.O. Box 243, Cockeysville, MD 21031) in a 50 ml flask.
  • the culture is incubated at 29°C in a gyrotory incubator overnight.
  • the FP43 stock solution is prepared by adding ⁇ 5 ml of NC broth to the plate showing nearly confluent lysis, incubating the plate at room temperature for one to two hours, and collecting the broth from the plate. The solution was centrifuged and the resulting super­natant passed through a 0.45 ⁇ filter to remove debris.
  • the resulting phage FP43 solution typically contains 109 to 1010 FP43 particles per ml--the exact titer is determined by plating several dilutions of the phage stock on a sensitive strain, such as S . griseofuscus C581.
  • Example 1B To prepare phage FP43 DNA, the procedure described in Example 1B was followed, except the lysates were prepared using four to six 9.5" x 9.5" Petri dishes. The plates were washed with 50 ml of NC broth to collect the phage particles. The lysates were centrifuged and the supernatants passed through a 0.45 ⁇ filter to remove cellular debris. The lysate was then centrifuged for 2 hours at 25°C at 30,000 rpm to pellet the phage particles. Each pellet was resuspended in 1 ml of ⁇ buffer; this solution was centrifuged in a tabletop centrifuge to pellet material that did not go back into solution.
  • the supernatant was then layered on top of a CsCl gradient composed as follows (from most dense to least dense): 750 ⁇ l of 1.7 ⁇ CsCl in ⁇ buffer; 750 ⁇ l of 1.6 ⁇ CsCl in ⁇ buffer; 750 ⁇ l of 1.4 ⁇ CsCl in ⁇ buffer; and ⁇ 2.45 ml of phage FP43 in ⁇ buffer.
  • the gradient was prepared in a polyallomer tube (1 ⁇ 2" x 1 ⁇ 2”), which was placed in an SW50.1 rotor (Beckman Instruments, Inc., Spinco Division, P.O. Box 10200, Palo Alto, CA 94304).
  • the solution was centrifuged at 25,000 rpm at 15°C for 1 hour and yielded two bands: a brown-tinged top band and a blue-tinged lower band.
  • the lower band was collected with a syringe and dialyzed against 2 to 3 liters of ⁇ buffer overnight.
  • the dialyzed band was extracted for 30 minutes with 1.5 volumes of ⁇ buffer-saturated phenol (25 ml phenol:10 ml ⁇ buffer) and then re-extracted for 10 minutes with another 1.5 volumes of ⁇ buffer-saturated phenol.
  • the phage band was then extracted 3 times with one volume of Sevag.
  • the precipitated phage FP43 DNA was spooled from the solution, washed with 70% ethanol and resuspended in ⁇ 1 ml of TE buffer, yielding a solution containing ⁇ 0.5 mg/ml of phage FP43 DNA.
  • a lyophilized culture of Streptomyces lividans /pIJ702 (ATCC 39155) is used to inoculate 10 ml of TSS medium containing 25 ⁇ g/ml thiostrepton. The culture is incubated at 29°C until the cells reach early stationary phase. The culture was then homogenized, and 5 ml of the homogenized culture were used to inoculate 100 ml of TSS also containing thiostrepton. The 100 ml of culture were incubated at 29°C until the Streptomyces lividans /pIJ702 cells reached stationary phase.
  • the cells were collected and washed once with a 10.3% sucrose solution.
  • the solution was mixed and then incubated at 30°C for 30-60 minutes, and then, about 18 ml of a solution that was 0.3 m NaOH, 1% SDS, and prewarmed to 50°C were added, mixed and the resulting mixture incubated at 80°C for 10 minutes.
  • the mixture was then cooled to room temperature, and 12 ml of a solution made by mixing 500 g phenol, 500 g CHCl3, and 0.5 g 8-hydroxyquinoline in 200 ml H2O were added and mixed well with the cell-­extract.
  • the phases were separated by centrifugation at 6000-8000 rpm for 10 minutes; approximately 45 ml of the resulting upper phase were transferred to a clean bottle.
  • the fraction con­taining the plasmid band was extracted 3-5 times with isopropanol saturated with TE buffer and CsCl to remove the ethidium bromide. After the extractions, the sample was diluted with four volumes of TE buffer, and then, two-and-one-half volumes of ethanol were added. The resulting solution was mixed and incubated overnight at -20°C.
  • the precipitate resulting from the overnight incubation at -20°C was collected by centrifugation (10,000 rpm for 30 minutes), dried, and reprecipitated twice.
  • the precipitations were done by suspending the pellet in TE buffer, adding NaOAc to 0.3 M, adding 2.5 volumes ethanol, chilling at -70°C for 10-15 minutes, and then centrifuging the solution as above.
  • the proce­dure yields about 100 ⁇ g of plasmit pIJ702 DNA, which was suspended in TE buffer at a concentration of 1 ⁇ g/ ⁇ l and stored at 4°C.
  • a restriction site and function map of plasmid pIJ702 is presented in Figure 1 of the accompanying drawings.
  • the mixture was then placed at 65°C, and another 10 ⁇ l of a 1:3 dilution of CAP were added to the solution which was incubated at 65°C for another30 minutes. Yet another 10 ⁇ l of a 1:3 dilution of CAP was again added to the solution, which was incubated at 65°C for another 30 minutes.
  • the Sph I-digested, CAP-treated plasmid pIJ702 DNA was extracted twice with ⁇ buffer-saturated phenol, extracted three times with ether, and collected by adjusting the sodium acetate (NaOAc) concentration of the reaction mixture to 0.30 M, adding two volumes of ethanol, chilling the reaction mixture to -70°C, and centrifuging to pellet the pre­cipitated DNA. The pellet was resuspended in 50 ⁇ l of TE buffer.
  • NaOAc sodium acetate
  • the ligated DNA constituted the desired plasmid pRHB101 DNA.
  • a restriction site and function map of plasmid pRHB101 is presented in Figure 2 of the accompanying drawings.
  • the ligated DNA after precipitation and resuspension in 10 ⁇ l of TE buffer, was used to transform Streptomyces lividans TK23 as described in Example 4, below.
  • Plasmid pRHB106 was constructed in accordance with the foregoing procedure except that plasmid pMT660 was used instead of plasmid pIJ702. However, for con­venience, plasmid pRHB106 can also be obtained in Streptomyces griseofuscus C581 from the NRRL under the accession number NRRL 18183.
  • Plasmid pRHB106 need not be isolated from a phage FP43 genomic library as is described for plasmid pRHB101 in Examples 2 and 4. Instead, S . griseofuscus C581/pRHB106 can be used to prepare a transducing lysate as described in Example 4. The transducing lysate will contain phage particles that have packaged plasmid pRHB106 DNA and can be used in transductions as described in Example 5.
  • Example 2 a genomic library of phage FP43 was con­structed, which included the hft -containing plasmid pRHB101.
  • the procedure set forth below demonstrates how plasmid pRHB101 was isolated from the genomic library. Briefly stated, the procedure first involves protoplast transformation of Streptomyces lividans TK23. These transformants, identified on the basis of their mel ⁇ , tsrR phenotype, were examined for size of insert (FP43) DNA.
  • the lysate that yielded the highest transduction frequency comprised plasmid pRHB101 packaged into phage particles.
  • Streptomyces lividans TK23 (NRRL 15826) was grown in a 10 ml culture for 40-48 hours at 30°C in TSS broth. The culture was then homogenized and sonicated, and the mycelial fragments were recovered by centri­fugation (800Xg for 10 minutes in a bench top centri­fuge) and washed once with 10 ml of P media. The mycelial fragments were resuspended in 10 ml of P media containing 5 to 10 mg/ml of egg-white lysozyme (Cal­biochem-Behring, P.O. Box 12087, San Diego, CA 92112) and incubated for 1 hour at 4°C.
  • the suspension was pipetted up and down once or twice to disperse clumps.
  • the protoplasts were recovered by centrifugation (800Xg for 10 minutes) and washed twice with 10 ml of p medium. The protoplasts were then suspended in 10 ml of P medium.
  • Thiostrepton-resistant, white transformants were islated, and a number of single colonies were used to inoculate 10 ml TSB cultures containing thiostrepton (25 ⁇ g/ml). The cultures were homogenized and then grown overnight at 30°C in a rotary shaker.
  • Plasmid isolation for analysis was in accordance with the procedures described in Example 2A; the CsCl gradients of Example 2A were replaced by ethanol precipitaions.
  • the mycelium was collected by centrifugation, washed twice with 10.3% sucrose, and then suspended in 1-2 ml of 10.3% sucrose.
  • Four hundred ⁇ l of the cell mixture were transferred to a small tube, and 100 ⁇ of lysozyme solution were added.
  • the sus­pension was incubated at 30°C for 30-60 minutes, fol­lowed by the addition and mixing of 300 ⁇ l of 0.3 M NaOH containing 1% SDS. The latter solution was kept at 50°C before its addition to the cell mix.
  • the cell mixture was placed at 80°C for 10 minutes, cooled to room temperature, and then extracted with 200 ⁇ l of phenol:CHCl3 (50:50).
  • the aqueous phase was transferred to a clean tube, made 0.3 M in NaOAc, and then one volume of isopropanol was added.
  • the DNA is incubated at room temperature for five minutes and then pelleted by centrifugation.
  • the pellet was dissolved in 400 ⁇ l of TE buffer and made 0.3 M in NaOAc. About 2.5 volumes of ethanol were added, and the mixture was incubated at -70°C for 30 minutes. After centrifugation and another precipitation, the plasmid DNA was suspended in 50 ⁇ l of TE buffer. Restriction enzyme cutting and electro­phoretic anlysis of the reaction products were used to determine plasmid structure.
  • a variety of different plasmids, each containing a different Sph I restriction fragment of phage FP43 were isolated by this procedure.
  • Example 4A The plasmids isolated in Example 4A were used to transform Streptomyces griseofuscus C581.
  • a 10 ml overnight culture of S . griseofuscus was prepared as described for S . lividans in Example 4A.
  • the culture was collected by centrifugation, washed with 3 ml of P media, and resuspended in 3 ml of P media containing 5 to 10 mg/ml of lysozyme.
  • the cells were then incu­bated at 4°C for one hour, collected by centrifugation, washed twice with 3 ml of P media, and resuspended in 3 ml of P media.
  • Example 4A For each plasmid prepared in Example 4A (about 0.5 ⁇ g in 10 ⁇ l of TE buffer), about 150 ⁇ l of protoplasts were added to the solution of plasmid DNA. Then, about 100 ⁇ l of 55% PEG 1000 and 1 ml of P media were added to each protoplast-DNA mixture. The cells were plated and overlayed with thiostrepton as described in Example 4A.
  • Example 4B For each plasmid, several transformants obtained in Example 4B were used to prepare lysates using phage FP43 as described in Example 1. These lysates were then used to transduce Streptomyces griseofuscus C581. Transduction was carried out by first obtaining an overnight culture of S . griseofuscus C581, homogenizing and sonicating that culture, and removing several 100 ⁇ l aliquots. To each aliquot was added 100 ⁇ l of one of the lysates, both directly and after serial dilution, and the mixture was plated on R2 agar using R2 overlays. Thiostrepton was added via an overlay at least 6 hours after plating. The lysate that yielded the greatest number of thiostrepton-­resistant transductants contained plasmid pRHB101 packaged into infective FP43 phage particles.
  • transducing Streptomyces griseofuscus described in Example 4C is generally applicable to the actinomycetes. Essentially, trans­duction merely requires mixing a culture of the organ­ism to be transduced with a transducing lysate and plating the resulting mixture. If FP43 antiserum is used in the transduction, the antiserum should be added about 3 to 6 hours after the cells are plated. It is convenient to add the antiserum to the plates using an overlay. If the antiserum is prepared in accordance with the procedure described in Example 6, only about 3 ⁇ l of the antiserum are added per transformation plate. It is convenient to add the thiostrepton to the plates in the same overlay that contains the antiserum.
  • All immunizations were carried out using a multiple (double) emulsion prepared from a water-in-­oil emulsion of the bacteriophage suspension. From 1.32 x 1010 to 3.44 x 1010 phage were administered to each rabbit in the multiple emulsion on each of six occasions throughout the course of the 16 week immuni­zation schedule.
  • Bacteriophage were supplied as suspensions containing from 1 to 1.6 x 1011 phage/ml. About 0.858 to 1.17 ml of these suspensions made up to at least 1.0 ml with sterile water were added to 1 ml of adjuvant. Dispersion of the aqueous phage suspension in oil emulsion was made using a Sorvall Omi-Mixer with a micro attachment. All procedures involved in making the multiple emulsion were carried out in an ice water bath. The primary emulsion was made at a speed setting of 5 for 5 minutes increasing to 6 for 4 minutes and finally 7 for 1 minute.
  • the primary disperse phase was checked by microscope to assure an even dispersion of small, uniform/sized droplets of the aqueous phage containing the phage within larger, uniformly sized oil droplets. Also, a small drop of the emulsion was placed on the surface of water. A satisfactory emulsion will not spread. This thick, creamy emulsion was then reemulsified with 2 ml of 2 percent Tween 80(poly­oxethylene sorbitan mono-oleate, Sigma) in saline. The resultant free flowing multiple emulsion was then used to immunize the rabbits.
  • the initial immunization was made using Bacto Adjuvant, complete H37Ra adjuvant (Difco Laboratories) containing 1 mg of killed and dried Mycobacterium tuberculosis H37Ra per ml of the adjuvant mixture of 15 percent arlacel A (mannide monooleate) and 85 percent bayol F (paraffin oil). All subsequent immunizations were made using Bacto adjuvant, incomplete Freund (Difco Laboratories) similar to the complete adjuvant but lacking the Mycobacterium .

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EP88301763A 1987-03-02 1988-03-01 Segment d'ADN conférant transduction à haute fréquence de vecteurs d'ADN recombinants Expired - Lifetime EP0281356B1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0341776A2 (fr) * 1988-05-09 1989-11-15 Merck & Co. Inc. Procédé d'incorporation des molécules d'ADN dans des microorganismes avec un système de restriction methyl-spécifique
EP0438232A2 (fr) * 1990-01-19 1991-07-24 Eli Lilly And Company Fragment de DNA, conférant un phénotype d'inhibition de plaque
EP0438231A2 (fr) * 1990-01-19 1991-07-24 Eli Lilly And Company Fragment de DNA, isolé à partir du phage FP43, fonctionnant comme une origine de réplication chez les streptomyces et genres voisins
US5194387A (en) * 1988-05-09 1993-03-16 Merck & Co., Inc. Process for the incorporation of DNA molecules into microorganisms with methyl-specific restriction systems
EP1041141A1 (fr) * 1999-03-30 2000-10-04 Council Of Scientific And Industrial Research Nouveau bactériophage, son procédé d'isolement et milieu de croissance universel utilisable dans le procédé
WO2000058482A2 (fr) * 1999-03-26 2000-10-05 The University Of Georgia Research Foundation, Inc. Phages transducteurs
US6482632B1 (en) 1999-03-29 2002-11-19 Council Of Scientic And Industrial Research Bacteriophage, a process for the isolation thereof, and a universal growth medium useful in the process thereof
WO2023057688A1 (fr) * 2021-10-06 2023-04-13 Turun Yliopisto Ingénierie métabolique d'actinomycètes par sélection de mutant de cellule unique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5974729A (en) * 1998-05-01 1999-11-02 Clark; Suzanne Fruit or vegetable guard

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Journal of Bacteriology, Vol. 159, No. 2, August 1984 (Baltimore, USA) K.L. COX et al. "Restriction of Bacteriophage Plaque Formation in Streptomyces Spp." pages 499-504 * Totality * *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5194387A (en) * 1988-05-09 1993-03-16 Merck & Co., Inc. Process for the incorporation of DNA molecules into microorganisms with methyl-specific restriction systems
EP0341776A3 (fr) * 1988-05-09 1990-07-25 Merck & Co. Inc. Procédé d'incorporation des molécules d'ADN dans des microorganismes avec un système de restriction methyl-spécifique
EP0341776A2 (fr) * 1988-05-09 1989-11-15 Merck & Co. Inc. Procédé d'incorporation des molécules d'ADN dans des microorganismes avec un système de restriction methyl-spécifique
EP0438231A2 (fr) * 1990-01-19 1991-07-24 Eli Lilly And Company Fragment de DNA, isolé à partir du phage FP43, fonctionnant comme une origine de réplication chez les streptomyces et genres voisins
EP0438231A3 (en) * 1990-01-19 1992-09-16 Eli Lilly And Company A dna segment isolated from phage fp43 functions as an origin of replication in streptomyces and related genera
EP0438232A3 (en) * 1990-01-19 1992-09-23 Eli Lilly And Company Dna sequence conferring a plaque inhibition phenotype
US5198360A (en) * 1990-01-19 1993-03-30 Eli Lilly And Company Dna sequence conferring a plaque inhibition phenotype
EP0438232A2 (fr) * 1990-01-19 1991-07-24 Eli Lilly And Company Fragment de DNA, conférant un phénotype d'inhibition de plaque
US6589732B2 (en) 1999-03-26 2003-07-08 The University Of Georgia Research Foundation, Inc. Transducing phages
US6696295B2 (en) 1999-03-26 2004-02-24 University Of Georgia Research Foundation, Inc. Transducing phages
WO2000058482A2 (fr) * 1999-03-26 2000-10-05 The University Of Georgia Research Foundation, Inc. Phages transducteurs
WO2000058482A3 (fr) * 1999-03-26 2001-02-15 Univ Georgia Res Found Phages transducteurs
US6245504B1 (en) 1999-03-26 2001-06-12 The University Of Georgia Research Foundation, Inc. Transducing phages of Actinomycetales
US6482632B1 (en) 1999-03-29 2002-11-19 Council Of Scientic And Industrial Research Bacteriophage, a process for the isolation thereof, and a universal growth medium useful in the process thereof
US6787360B2 (en) 1999-03-29 2004-09-07 Council Of Scientific And Industrial Research Bacteriophage, a process for the isolation thereof, and a universal growth medium useful in the process thereof
EP1041141A1 (fr) * 1999-03-30 2000-10-04 Council Of Scientific And Industrial Research Nouveau bactériophage, son procédé d'isolement et milieu de croissance universel utilisable dans le procédé
WO2023057688A1 (fr) * 2021-10-06 2023-04-13 Turun Yliopisto Ingénierie métabolique d'actinomycètes par sélection de mutant de cellule unique

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CA1318620C (fr) 1993-06-01
DE3886238D1 (de) 1994-01-27
IL85496A0 (en) 1988-07-31
AU1232688A (en) 1988-09-01
IE62222B1 (en) 1995-01-11
DK95188A (da) 1988-12-16
HU209151B (en) 1994-03-28
AU605249B2 (en) 1991-01-10
DE3886238T2 (de) 1994-04-28
IL85496A (en) 1992-11-15
HUT46358A (en) 1988-10-28
ES2048199T3 (es) 1994-03-16

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